JP2014063151A - Microscope illumination optical system and microscope having the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an illumination optical system suitable for a three dimensional fluorescence microscope which provides a high-quality sectioning effect without having to require scanning of an object surface or a precisely controlled illumination system.SOLUTION: An illumination optical system of a microscope for observing a sample placed on an object plane 101 illuminates the object plane. A plurality of coherent light source regions A, B, C are arranged spaced apart from each other on a pupil plane of the illumination optical system such that at least one of distances dA, dB, dC from the centers cA, cB, cC of the plurality of light source regions to the center cP of the pupil of the illumination optical system is different from the others.

Description

  The present invention relates to a microscope illumination optical system that illuminates a sample in a microscope, and more particularly to a microscope illumination optical system suitable for a three-dimensional fluorescence microscope.

  Observation of biological samples with a microscope, in particular a fluorescence microscope, is essential for biological research including medical applications. However, when a thick sample is observed with a normal fluorescence microscope, an image in which images at all height positions where light inside the sample is transmitted is superimposed is observed. That is, in addition to the image of the plane at the height position where the focus is in focus (focusing plane), the blur image of the plane at the height position where the focus is out of focus (non-focusing plane) is superimposed and observed. As described above, in an ordinary fluorescent microscope, it is not possible to selectively separate only an image on a desired focal plane. The effect of selectively separating and extracting only the image of the desired in-focus plane is called a “sectioning effect”.

  A fluorescence microscope configured to obtain a sectioning effect based on various mechanisms is called a three-dimensional fluorescence microscope and is distinguished from a normal fluorescence microscope. When there is a sectioning effect, it is possible to create a three-dimensional three-dimensional image by laminating images of arbitrary focal planes on a computer. That is, anyone can perform the stereoscopic view of the cell arrangement that has been performed in the brain by an experienced pathologist or the like by digital processing.

  As a typical three-dimensional fluorescence microscope, there is a confocal microscope. This is a system that blocks only light coming from the desired in-focus plane and shields light with a low degree of convergence coming from the out-of-focus plane by placing a pinhole at the condensing point of the light coming from the in-focus plane. It is. This method has a high sectioning effect, but since the area that can be imaged at one time is narrow in a dot shape, scanning is necessary to observe the entire area of the sample.

  On the other hand, there is a structured illumination method as a method of realizing a sectioning effect by using image processing by a computer (see Non-Patent Document 1). In this method, a situation is created in which the illumination intensity changes, for example, in a sine wave shape on the object plane, and a plurality of images in which the sine wave structure is translated are acquired by shifting the phase. Thereafter, the sectioning effect is obtained by processing the plurality of images on the computer. This scheme requires the creation of a sinusoidal structure with controlled phase, ie position, with high accuracy.

  Furthermore, a method of using randomly generated speckle as illumination is also known (see Patent Document 1 and Non-Patent Documents 2 to 6). This method also uses image processing by a computer. However, since the illumination intensity on the object surface depends on random speckles, there is a disadvantage that uneven intensity unevenness is unavoidable in the final image.

US Patent Publication No. 2010/0224796

M. A. A. Neil and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22, 1905 (1997). C. Ventalon and J. Mertz, “Quasi-confocal fluorescence sectioning with dynamic speckle illumination,” Opt. Lett. 30, 3350-3352 (2005). C. Ventalon and J. Mertz, “Dynamic speckle illumination microscopy with translated versus randomized speckle patterns,” Opt. Express 14, 7198-7209 (2006). C. Ventalon, R. Heintzmann, and J. Mertz, “Dynamic speckle illumination microscopy with wavelet prefiltering,” Opt. Lett. 32, 1417-1419 (2007). Daryl Lim, Kengyeh K. Chu, and Jerome Mertz, “Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy,” Opt. Lett. 33, 1819-1821 (2008). Daryl Lim, N. Ford, Kengyeh K. Chu, and Jerome Mertz, “Optically sectioned in vivo imaging with speckle illumination HiLo microscopy” Journal of Biomedical Optics. 16, 016014 (2011).

  From the above, it is desired to develop a three-dimensional fluorescence microscope that can obtain a high-quality sectioning effect without requiring scanning of an object plane or a highly controlled illumination system.

  The present invention provides an illumination optical system particularly suitable for realizing such a three-dimensional fluorescence microscope.

  An illumination optical system according to an aspect of the present invention is an illumination optical system that illuminates an object surface in a microscope that observes a sample disposed on the object surface, and a plurality of coherent lenses on the pupil plane of the illumination optical system. The light source regions are arranged apart from each other, and at least one of the distances between the centers of the plurality of light source regions and the center of the pupil of the illumination optical system is different from the other distances.

  Note that a microscope having the microscope illumination optical system and an objective optical system for observing the sample illuminated by the microscope illumination optical system also constitutes another aspect of the present invention.

  By using the illumination optical system of the present invention, it is possible to realize a three-dimensional fluorescence microscope capable of obtaining a high-quality sectioning effect without requiring high precision with respect to scanning of the object surface and the illumination system.

It is a figure showing the object O illuminated uniformly. It is a figure showing the object O by which speckle illumination was carried out. It is a figure showing the difference of FIG. 1 and FIG. It is a figure which represents typically the area | region which calculates the spatial dispersion value of the intensity | strength in the vicinity of the point (x, y) in FIG. It is a figure showing the non-uniformity of xy direction of (sigma) (x, y, z). (A) is a figure showing the example of the pupil function for implement | achieving the illumination light intensity distribution of a comb function shape, (b) is a figure showing the actual illumination light intensity distribution which can be performed when the pupil function is used. is there. It is a figure which shows the illumination light intensity distribution which can be made into the plane of z = +/- 2micrometer in a sample when the illumination optical system with a pupil function of Fig.6 (a) is used. It is a figure which shows the fluorescence intensity distribution observed in the plane of z = 0 micrometer in a sample, when illuminating the object O2 using the illumination optical system with a pupil function of Fig.6 (a). (A) is a figure which shows the pupil function P2 in Example 1 of this invention, (b) shows the illumination light intensity distribution in the plane of z = 0 micrometer in a sample when the illumination optical system which has this P2 is used. FIG. It is a figure which shows the fluorescence intensity distribution observed in the plane of z = 0 micrometer in a sample, when the object O2 is illuminated using the illumination optical system with a pupil function of Fig.9 (a). It is a schematic diagram which shows the example of arrangement | positioning in the three-dimensional fluorescence microscope of the illumination optical system of the Example of this invention. It is a figure showing the image of the object O2 obtained by the method of using the random speckle of a nonpatent literature 5 and 6 for illumination. It is a figure showing the image of the object O2 at the time of using the grating | lattice illumination which the illumination optical system provided with the pupil function P2 in Example 1 of this invention forms. It is a figure which shows the illumination light intensity distribution in the plane of z = 0 micrometer in a sample when the pupil function which has two light source points shown in Example 2 of this invention and the illumination optical system which has the pupil function is used. . It is a figure showing the image of the object O2 at the time of using the striped illumination which the illumination optical system provided with the pupil function with the two light source points shown in Example 2 forms. It is a figure explaining the pupil function of an Example. It is the schematic of a normal microscope. It is the schematic of the 1st Example of a lighting unit. It is a figure which shows the structure which uses three light beams in the 1st Example of an illumination unit. FIG. 6 is a schematic view of a second embodiment of the lighting unit. FIG. 6 is a schematic view of a third embodiment of the lighting unit. 6 is a schematic view of a light shielding member of Example 4. FIG. 10 is a schematic diagram of Example 5. FIG.

  Embodiments of the present invention will be described below with reference to the drawings.

  The illumination optical system for a microscope that is an embodiment of the present invention can be applied to, for example, a three-dimensional microscope that observes a sample as a self-luminous body whose light emission mechanism is fluorescence or phosphorescence. As the type of the microscope, either an episcopic microscope or a transmission microscope may be used.

  As a specific example, the illumination optical system for a microscope according to the embodiment can be applied to a microscope for observing a fluorescently stained sample that is a test sample used in a digital slide scanner. A digital slide scanner is a device that scans a preparation used in biology or pathological examination at high speed and converts it into high-resolution digital data. Furthermore, the illumination optical system for a microscope according to the embodiment can be used as a means for imparting a sectioning effect to, for example, a digital slide scanner including a projection optical system having a large numerical aperture (NA) or a normal fluorescence microscope.

  First, before describing in detail the illumination optical system for a microscope of the embodiment, problems of a method using a speckle that has been conventionally used will be described.

  In Non-Patent Documents 5 and 6, using only two images, an image 1 illuminated with uniform intensity and an image 2 illuminated with speckle, only the image of the fluorescent object in the in-focus plane is extracted. A method is disclosed. According to this, first, an image 3 showing an intensity difference between the image 1 and the image 2 is created by a computer. In order to illuminate an object with speckles, an object that causes random phase disturbance such as ground glass may be inserted into the pupil of an illumination optical system having coherent excitation light as a light source. Here, in order to simplify the description, let us consider a case where O (x, y, z) = δ (z), where O (x, y, z) is the intensity distribution of the fluorescent object. In the following description, the intensity distribution O (x, y, z) of the fluorescent object may be abbreviated as the object O. The object O is a virtual object that exists locally only in the plane of z = 0 and has a uniform intensity distribution in the xy direction. Then, z = 0 is set as a focusing plane. Further, a plane with z = ± a (a> 0) is a representative of the out-of-focus plane.

  FIG. 1A shows an image obtained when the object O is illuminated with illumination having a uniform intensity distribution, and is observed while being focused on the in-focus plane. Let Is (x, y, z) be an image taken with illumination having this uniform intensity distribution. Further, FIG. 1B is an image obtained when observation is performed with the same illumination focused on a non-focused plane. These images correspond to the image 1 described above.

  FIG. 2A shows an image obtained when the object O is illuminated with speckles and observed with the focus plane in focus. Further, FIG. 2B is an image obtained when observation is performed with the same illumination focused on a non-focused plane. These images correspond to the image 2 described above.

  Further, FIG. 3A is an image showing a difference between the intensity distribution of the image shown in FIG. 1A and the intensity distribution of the image shown in FIG. FIG. 3B is an image showing a difference between the intensity distribution of the image shown in FIG. 1B and the intensity distribution of the image shown in FIG. These images correspond to the image 3 described above.

  As is clear from FIGS. 1A and 1B, an image obtained by focusing on z = 0 where the object O actually exists and the original object when the image is taken using normal uniform illumination. The image obtained when focusing on z = ± a (a> 0) where no O exists is exactly the same and cannot be distinguished. Thereby, it turns out that there is no sectioning effect in a normal fluorescence microscope.

  Non-Patent Documents 5 and 6 disclose a method of extracting data reflecting the intensity distribution O of an actual fluorescent object from these data. Specifically, the images shown in FIGS. 3A and 3B are taken into the computer, and the spatial dispersion of the intensity difference in the region near the point (x, y) (the region indicated by the white frame in FIG. 4). Calculate the value σ. Then, a map σ (x, y, z) of the dispersion values obtained in this way is created. At this time, as can be easily imagined from FIGS. 3A and 3B, the black and white contrast is sharp in the image of FIG. 3A corresponding to the image obtained by processing the light from the in-focus plane. , Σ (x, y, 0) is a high value. On the other hand, the image shown in FIG. 3B corresponding to the image obtained by processing the light from the out-of-focus plane has little contrast due to the blur of the speckle image. For this reason, σ (x, y, a) is almost uniformly close to zero.

Therefore, if I (x, y, z) is calculated using the following equation (1), I (x, y, z) becomes an image in which the sectioning effect is obtained by σ (x, y, z). . That is, I (x, y, 0) has a value, but I (x, y, a) has almost no value.
I (x, y, z) = Iu (x, y, z) · σ (x, y, z). . . (1)
Here, Iu (x, y, z) is an image picked up using normal uniform illumination so that the position at the height z is in focus.

  In this way, an image close to the actual object O can be reconstructed on the computer. However, this method has an inevitable drawback because it uses a speckle phenomenon, which is essentially a random phenomenon, as illumination. Hereinafter, this defect will be described.

  Originally expected I (x, y, 0) is Iu (x, y, 0) homogeneous in the xy direction as shown in FIG. That is, σ (x, y, 0) is also expected to be uniform in the xy direction. However, in practice, FIG. 5 shows that σ (x, y, 0) is never homogeneous in the xy direction. This is so-called illumination unevenness caused by the speckles not having a uniform distribution, and this greatly degrades the final image quality of I (x, y, 0).

  Therefore, in this embodiment, an illumination method having a sectioning effect is provided while preventing image quality deterioration due to illumination unevenness. Hereinafter, the principle will be described.

  This example is based on the following mathematical facts. In general, the function represented by Expression (2) is referred to as a comb function.

comb (x, y) = Σδ (x−mp) δ (y−np). . . (2)
Here, δ represents a Dirac delta function, and p is an interval (pitch) in a direction along the coordinate axis between points having infinite values. The sum symbol is an integer of −∞ <m and n <∞.

  The mathematical fact about this comb function is that its Fourier transform is also a comb function with a pitch of 1 / p, as shown in equation (3).

F [comb (x, y)] (f, g) = Σδ (fm−p) δ (gn−p). . . (3)
Here, F is a symbol representing Fourier transform, and f and g represent spatial frequencies corresponding to x and y, respectively.

  In general, an amplitude distribution on the image plane is a Fourier transform of the amplitude distribution P (f, g) (pupil function) in the pupil of the optical system. When the optical system is an illumination optical system, the square of the absolute value of the amplitude distribution on the image plane is the intensity distribution of the light that illuminates the sample object. Therefore, if P (f, g) of the illumination optical system is set to the comb function, it is expected that the illumination light having the comb function is realized. Since the comb-function illumination light is a light with a uniform intensity distributed at a uniform pitch on the object surface, it is expected that no illumination unevenness will occur.

  Non-Patent Documents 5 and 6 describe that the calculation area of σ shown in FIG. 4 can be reduced when the pitch of illumination light on the object plane is as fine as possible, so that the resolution performance in the horizontal direction is improved. ing. Therefore, the pitch on the pupil plane of the illumination optical system should be as large as possible. Actually, since the pupil of the optical system has only a finite size, it is impossible to realize illumination that continues infinitely in the gg direction as expressed by the equation (3). However, even if only the minimum constituent unit of the comb function is adopted as the amplitude distribution on the pupil plane, illumination light without illumination unevenness can be realized.

This is shown using FIG. FIG. 6A shows illumination in which the minimum square appearing in the comb function expressed by Expression (2) is adopted as P (f, g). FIG. 6B shows the illumination intensity that can be formed on the object surface at that time. Here, the used wavelength λ = 500 nm and the numerical aperture NA = 0.7. The pupil of the illumination optical system uses a standardized radius of 1, and the coordinates of the position having the amplitude at this time are
(1 / √2, 1 / √2)
(-1 / √2, 1 / √2)
(-1 / √2, -1 / √2)
(1 / √2, -1 / √2)
It is.

  If the method for calculating σ (x, y, 0) described above is used for an object illuminated with periodic illumination light as shown in FIG. 6B, there is no illumination unevenness, so the uniformity is very high. σ (x, y, 0) is obtained.

  However, the illumination provided as shown in FIG. It is known that when such a pupil function P (f, g) of the illumination optical system is used, the illumination distribution at a place away from the image plane (that is, the object plane) is hardly blurred. This is shown in FIG.

  FIG. 7 shows the distribution of illumination light at a position where z = ± 2.0 μm. When FIG. 7 is compared with FIG. 6B, it can be seen that almost no blur can be confirmed. In order to explain the situation that causes a problem under such circumstances, consider the object O2 (x, y, z) = δ (z + 1) + δ (z−1). The unit of z is μm.

  When imaging the object O2, of course, it should not have intensity at z = 0. If it has intensity, it needs to be much lower than the intensity of the image at z = ± 1.

  Now, assume that the fluorescent object located at z = 1 on the upper side of the object O2 and the fluorescent object located at z = -1 on the lower side are illuminated with substantially the same illumination shape. However, the illumination given as shown in FIG. 6A has a small amount of blur. For this reason, the comb function-like fluorescence coming from the upper fluorescent object and the comb function-like fluorescence coming from the lower fluorescent object exactly overlap each other at the position of z = 0 to form a light intensity distribution with very high contrast. . The light intensity distribution formed at z = 0 is shown in FIG. As described above, the high contrast state keeps the value of σ (x, y, 0) high. As a result, an image as if the fluorescent object exists at the position of z = 0 where the object O2 should not exist is obtained.

Therefore, in this embodiment, P2 (f, g) shown in FIG. 9A is used as a pupil function (amplitude distribution) P (f, g) on the pupil plane of the illumination optical system for solving this problem. Is used. P2 (f, g) is a triangular coordinate similar to a triangle formed by three points in the pupil indicated by the following coordinates or a triangle formed by the three points in the orthogonal coordinate system on the pupil plane normalized by the radius 1 of the illumination optical system. It is characterized in that point light sources coherent with each other are arranged at three vertices.
(-1 / √2 + a, 1 / √2 + b)
(-1 / √2 + a, -1 / √2 + b)
(1 / √2 + b, -1 / √2 + a)
A and b in these coordinates are real numbers.

  In other words, in the pupil function P2 (f, g), as shown in FIG. 16, a plurality of light source regions A, B, and C that are coherent with each other are arranged apart from each other on the pupil plane of the illumination optical system. At least one of the distances dA, dB, dC between the centers cA, cB, cC of the light source regions A, B, C and the center cP of the pupil of the illumination optical system is another distance. And different. The light source region here includes a point light source that can be regarded as having a minute region. The light source region is preferably a region having a size ratio of less than 0.3 with respect to the radius of the pupil. That at least one distance is different from other distances means that all distances may be different, or that only one distance may be different from two or more other same distances.

  FIG. 9B shows a grid-like illumination light intensity distribution on the object plane that can be actually obtained using the pupil function (amplitude distribution) P2 (f, g). A right-angled isosceles triangle formed by connecting three points having amplitudes on the pupil plane is also a repetitive unit of the comb function, so that the formed illumination shape is similar to the illumination intensity shown in FIG. The shape of each spot is slightly elliptical, but there is no illumination unevenness. Thus, the effect of P2 in which a plurality of coherent light sources are intentionally arranged asymmetrically with respect to the origin (center of the pupil) is that the illumination light is obliquely incident, and therefore the intensity distribution of the illumination light is laterally shifted when z changes. It appears in.

  In order to verify this effect, the fluorescent object located at z = 1 on the upper side of the object O2 and the fluorescent object located at z = -1 on the lower side are again illuminated with their positions shifted by the illumination formed by P2. Think about the situation. In this situation, the fluorescence coming from the upper fluorescent object and the displaced fluorescent light coming from the lower fluorescent object at the position of z = 0 do not overlap exactly, but overlap and shift so that the light intensity with very low contrast A distribution is formed. This is shown in FIG. When FIG. 10 is compared with FIG. 8, the image in FIG. 10 is a lower contrast image. Therefore, according to the present embodiment, it can be seen that it is possible to prevent the region where the original object does not exist near z = 0 from being inadvertently resolved.

  According to the illumination method (that is, the illumination optical system) of the present embodiment described above, when used in combination with the methods disclosed in Non-Patent Documents 5 and 6, a good image without intensity unevenness can be obtained for a long time. Can be obtained without scanning.

  Next, a preferred arrangement example of the illumination optical system of the present embodiment in the three-dimensional fluorescence microscope will be described with reference to FIG. In FIG. 11, reference numeral 100 denotes an epi-illumination type three-dimensional fluorescence microscope.

  Reference numeral 110 denotes an illumination optical system, which has a configuration that can be added to a microscope main body constituted by an objective lens 102 and an image sensor (imaging device) 103. Reference numeral 101 denotes an object (sample) arranged on the object plane.

  In the illumination optical system 110, reference numeral 111 denotes a coherent light source, and a laser having a wavelength that can excite a fluorescent sample can be used. Reference numeral 112 denotes an optical element such as a diffraction grating, a prism, or an optical fiber, which has an action of dividing one beam emitted from the light source 111 into a plurality of (for example, three) beams. The optical element 112 is not limited to the diffraction grating, the prism, or the like, and any optical element can be used as long as it can realize the pupil function shape characterized by the present embodiment on the pupil plane 113 of the illumination optical system 110. The pupil function shape forming method in the present embodiment can be realized by a method that is easy for an engineer related to a microscope or a semiconductor exposure apparatus, and for example, a computer-generated hologram (CGH) can be used.

  The divided beams pass through the objective lens (objective optical system) 102 via the dichroic mirror 114, and illuminate the object 101 with a grid-like illumination light intensity distribution. The fluorescence emitted from the object 101 passes through the objective lens 102, passes through the dichroic mirror 114, passes through the objective lens 102, and forms an image on the image sensor 103. An image obtained by imaging by the image sensor 103 and displayed on a monitor (not shown) is observed by an observer.

  In the above description, the number of point light sources on the pupil plane of the illumination optical system is three, but the number of point light sources is not limited to three. When the number of point light sources is three, the object 101 is irradiated with three light beams having incident angles corresponding to the positions of the respective point light sources, and a lattice-like pattern as shown in FIG. 9b is generated. As will be described in more detail in the second embodiment, even if the number of point light sources is two, the method of the present invention can be implemented without problems.

  When two point light sources are employed as the amplitude distribution P (f, g) on the pupil plane of the illumination optical system, a striped pattern as shown in FIG. As shown in FIG. 14A, since the two point light sources are arranged asymmetrically with respect to the pupil center, the intensity distribution of the striped pattern is laterally shifted when the focus coordinate z changes. This is the same as the case of the three asymmetric point light sources already described. Therefore, even when two point light sources that are not symmetrical with respect to the center of the pupil are set, a good sectioning image without intensity unevenness can be obtained as in the case of three asymmetric point light sources.

  Hereinafter, a plurality of coherent light source regions are arranged apart from each other on the pupil plane of the illumination optical system, and at least one of the distances between the centers of the plurality of light source regions and the pupil center of the illumination optical system is The intensity pattern of illumination light obtained by being different from other distances is collectively referred to as asymmetric structure illumination.

  The outline of the embodiment of the present invention has been described above. By applying the present invention, a three-dimensional fluorescence having a high-quality sectioning effect can be obtained by attaching an illumination unit for realizing both asymmetric structure illumination and illumination having a uniform intensity distribution to an ordinary fluorescence microscope. A microscope system can be constructed. In addition, the illumination unit can be realized simply by applying a modification that can be restored to its original state to an ordinary fluorescent microscope. Hereinafter, specific embodiments of the lighting unit will be described.

  In order to realize a three-dimensional fluorescence microscope, (a) an image 1 of a fluorescent sample when illuminated with excitation light having a substantially uniform intensity distribution (hereinafter sometimes simply referred to as uniform illumination) and ( b) It is necessary to take and acquire images 2 of the fluorescent sample by asymmetric structure illumination.

  First, the configuration and means of a lighting unit that realizes asymmetric structure lighting will be described. It is assumed that a fluorescent image is acquired using an image sensor such as a CCD, and an ordinary fluorescent microscope generally has a plurality of camera ports. Therefore, in the following description, it is assumed that there are a plurality of camera ports. To do. Further, since the eyepiece observation system does not play an essential role in the following description, the description and explanation in the figure are omitted.

  FIG. 17 is a schematic structural view of a normal fluorescence microscope. In the following description, common members are denoted by common numbers. Different members having the same function may be given the same number.

  Excitation light 301 (dotted line) from an excitation light source (not shown) such as a mercury lamp or a laser is guided into a normal fluorescence microscope 200 and becomes a light beam having a predetermined wavelength range through the excitation light filter 201. The dichroic mirror 114 is reached. The dichroic mirror 114 reflects light having a shorter wavelength than the wavelength approximately halfway between the excitation wavelength and the fluorescence wavelength of the fluorescent sample, and transmits light having a longer wavelength. Therefore, the excitation light 301 is reflected by the dichroic mirror 114, and is irradiated on the object 101 through the objective lens 102-A. The fluorescence 302 (solid line) emitted from the object 101 and the excitation light 303 (dashed line) reflected and scattered by the object 101 reach the dichroic mirror 114 through the objective lens 102-A. Although most of the fluorescence 302 is transmitted through the dichroic mirror 114 and reaches the fluorescence filter 203, most of the excitation light 303 reflected and scattered by the object 101 is reflected.

  The three members of the excitation light filter 201, the dichroic mirror 114, and the fluorescence filter 203 are configured as one unit with three bodies, and are almost detachable and replaceable by rotating with a turret or the like. .

  The fluorescent light 302 transmitted through the fluorescent filter 203 passes through the folding mirror 202, passes through the imaging lens 102-B, and is divided into two optical paths by the half mirror 204. A part of the light is guided to the first camera port 211 and forms an image on an image sensor 103 such as a CCD installed there. The rest reaches the second camera port 212. The first and second camera ports 211 and 212 are provided at positions conjugate with the object 101, and the image plane of the image sensor 103 is installed on a plane conjugate with the object 101. A surface optically conjugate with the object 101 inside the first and second camera ports 211 and 212 is hereinafter referred to as an in-camera port conjugate surface.

  Most modern microscopes, including ordinary fluorescence microscopes, employ the infinity correction method, and the fluorescence from the sample is converted into a parallel beam by the objective lens and propagates in the form of a parallel beam to the imaging lens. It is a mechanism to collect light. In general, a microscope is a telecentric optical system on both the image side and the object side. Under the above conditions, in a microscope employing the infinity correction method, the sample is focused when positioned at the front focal point of the objective lens, and forms an image at the rear focal point of the imaging lens. The position of the rear focal point of the objective lens and the front focal point of the imaging lens coincide with each other. The position in the optical axis direction of the conjugate plane in the camera port and the rear focal point of the imaging lens coincide.

  In order to realize asymmetrical illumination on an object, it is necessary to divide the excitation light into a plurality of coherent light beams having a predetermined incident angle, and to create an interference region where the light beams overlap on the object surface. is there. Hereinafter, the coherent light beams may be simply referred to as a light beam. When realizing such asymmetrical structure illumination with respect to an existing ordinary fluorescence microscope, it becomes a problem where excitation light is incident from. However, in the present invention, as described above, in an ordinary fluorescence microscope having a camera port, Note that the conjugate plane in the camera port and the object plane have a conjugate relationship.

In particular, it is assumed that the image sensor 103 such as a CCD is disposed in the first camera port 211 and the excitation light divided into a plurality of light beams for asymmetric structure illumination is incident from the second camera port 212. Then, the excitation light divided into the plurality of light fluxes enters the microscope from the second camera port 212 and forms an image of a normal object such as the imaging lens 102-B, the objective lens 102-A, and the object 101. The sample can be irradiated with excitation light by following the reverse optical path. Since the camera port and the sample are in a conjugate position, if a plurality of incident light beams overlap on the conjugate plane in the camera port, they also overlap on the object plane and interfere with each other to realize asymmetrical illumination. It is guaranteed.
The pitch of the asymmetric structure illumination pattern is defined by the incident angles of a plurality of light beams on the object plane. These angles can be defined by angles formed with respect to the optical axes of a plurality of light beams incident on the camera port. Specifically, when the imaging magnification of the object with respect to the conjugate plane in the camera port is m, and the incident angle of each light beam on the object surface is θ ₂ , the angle θ formed with respect to the optical axis of the light beam incident on the camera port ₁ is
sin θ ₁ = sin θ ₂ / m
You can decide to satisfy.

  Next, the optical properties of the incident excitation light will be described. Here, since the light source used for the asymmetric structure illumination needs to be coherent, a laser light source may be used. As the laser light source, it is possible to use a semiconductor laser, a gas laser, or the like having an oscillation wavelength in the excitation wavelength region. One important concept for laser light is the beam waist. The beam waist is a position where the beam diameter of the laser beam is minimized, and the beam diameter of the laser beam is larger before and after propagation than the beam diameter at the beam waist. Further, it is known that the radius of curvature of the wavefront of the laser beam becomes maximum (plane) at the beam waist. In order to reduce distortion of the intensity pattern of the asymmetric structure illumination, it is desirable that each wavefront of the plurality of light beams is flat on the object plane. Therefore, the plurality of light beams for forming the asymmetric structure illumination may be composed of a plurality of laser beams that respectively form beam waists on the object plane. In the above, the method of realizing asymmetric structure illumination using a normal fluorescence microscope and preferable conditions have been described.

  Next, the configuration of an illumination unit that produces a plurality of light beams and the modifications necessary to attach the illumination unit to a normal fluorescence microscope will be described.

  FIG. 18 is a schematic diagram of a three-dimensional microscope system configured by attaching an illumination unit for asymmetric structure illumination to a normal fluorescence microscope. For the eyepiece observation system, description and explanation in the figure are omitted. FIG. 18 exemplifies a case where a plurality of light beams are composed of two light beams and a striped pattern is projected onto the object plane.

  Reference numeral 400 denotes an illumination unit, which is connected to the normal fluorescence microscope 200 by a mount member 500 at the second camera port 212. Within the illumination unit 400 is a laser light source 111 for exciting the fluorescent sample, which emits a laser beam 301 (dotted line). The laser beam 301 enters the first optical path adjustment system 410. The optical path adjustment system 410 includes bending mirrors 411 to 414, and the laser beam 301 is reflected by the four bending mirrors in this order and enters the condenser lens 401. The laser beam 301 that has passed through the condenser lens 401 and the collimator lens 402 is incident on the second optical path adjustment system 420.

  The optical path adjustment system 420 includes bending mirrors 421 to 424, and the laser beam 301 is reflected by the four bending mirrors in this order and then enters the Mach-Zehnder optical system 430. The laser beam 301 incident on the Mach-Zehnder optical system 430 is divided into two laser beams 301-A and 301-B by intensity division by a half mirror 431. The laser beam 301 -A is reflected by the bending mirror 433 and reaches the half mirror 434. On the other hand, the laser beam 301 -B is reflected by the bending mirror 432 and reaches the half mirror 434. The intensity of both laser beams 301 is halved by the half mirror 434. Thereafter, the laser beam 301 enters the normal fluorescence microscope 200 through the mount member 500 and the second camera port 212.

  Thereafter, the two laser beams 301-A and 301-B reach the object 101 through the half mirror 204, the imaging lens 102-B, the bending mirror 202, and the objective lens 102-A. Here, the half mirror 431, the bending mirror 432, the bending mirror 433, and the half mirror 434 in the Mach-Zehnder optical system 430 each have an angle adjustment mechanism (not shown). The angle adjusting mechanism can be adjusted so that the two laser beams 301-A and 301-B of the Mach-Zehnder optical system overlap at the second camera port 212, and each irradiates a predetermined incident angle object surface.

  Hereafter, description will be given of a suitable beam diameter of the beam waist formed on the object 101 and setting of parameters of the optical system for realizing the beam diameter.

The beam diameter on the object 101 defines the illumination area on the object 101 and needs to be large enough to cover the observation area. When the focal length of the objective lens 102-A is f _obj and the focal length of the imaging lens 102-B is f _tube , the imaging magnification m from the object 101 to the image sensor 103 is expressed by m = f _tube / f _obj. . 1/2 of the length of the diagonal of the effective image pickup area of the image sensor 103 and W _image, when 1/2 of the length of the diagonal of the effective image pickup area of the on the object 101 and _{W obj W obj = W image} / M. Therefore, it is necessary to set the beam diameter on the object 101 to W _image / m or more. Hereinafter, the beam diameter on the object 101 will be described as W _image / m.

The position of the beam waist and the beam diameter at the beam waist can be converted by passing the lens. The beam widths (1 / e ² radii) at the beam waist before and after the lens transmission are w ₁ and w ₂ , respectively, and the distances from the beam waist to the lens before and after the lens transmission are d ₁ and d ₂ , respectively. Further, if the focal length of the lens is f and the wavelength of the laser is λ, the relationship of equation (4) is established. w ² = w ₁ ² · f ² / {(f−d ₁ ) ² + (π · w ₁ ² / λ) ² }
d ₂ = f + (w ₂ / w ₁ ) ² · (d ₁ −f) (4)
Further, the beam diameter at a position separated by a distance z from the beam waist of the laser beam having the beam waist w _o is w (z), the radius of curvature R (z) of the beam wavefront at that position, and sufficiently separated from the beam waist. If the beam divergence angle (half angle) at the position is θ _wo , the relationships of equations (5) and (6) are established.

w (z) ² = w _o ² {1+ (z · λ / (π · w _o ² )) ² }}
R (z) = z · {1+ (π · w _o ² / (z · λ)) ² } (5)
θ _wo = λ / (π · w _o ) (6)
Let us consider a case where a beam waist is placed at the front focal position of the lens and is incident on the lens using Expression (4). This corresponds to placing a _d 1 = f, the second expression of Expression (4) _to obtain _d 2 = f. Therefore, it can be seen that the beam waist position after passing through the lens coincides with the rear focal position of the lens. Further, when d ₁ = f in the first expression of the expression (4), it can be seen that there is a relationship of w ₁ · w ₂ = f · λ / π when deformed.

From the above, in order to set the beam diameter at the beam waist on the object 101 to W _obj , the beam diameter at the beam waist generated at the front focal point of the objective lens 102 -A is set as W _obj-front .
W _obj−front = f _obj / · λ / (π · W _obj )
Must be satisfied.

Similarly, the beam waist diameter W _{port2 at} the second camera port 212 is
W _port2 = f _tube · λ / (π · W _obj−front )
It is prescribed by.

Substituting the original W _obj-front formula here,
W _port2 = f _tube · λ / (π · f _obj · λ / (π · W _obj ))
= W _obj · f _tube / f _obj = W _obj · m = W _image
It becomes.

Thus, based on the illumination area required on the object plane 101, the beam diameter to be achieved at each beam waist in order to realize the illumination area can be determined using Equation (4). As can be seen from the above equation, the beam diameter W _port2 at the second camera port 212 does not change even when the imaging magnification m is changed.

  The beam waist propagation will be further described. The laser beam 301 has a beam waist diameter at the light emitting portion of the laser light source 111. Laser light sources have different intrinsic beam waist diameters and beam divergence angles for each light source. The relationship between the beam waist diameter, wavefront, and divergence angle is determined by the equations (4) to (6) already described. Specifically, when the beam waist is about submillimeter to millimeter as in many gas lasers, the beam opening angle is in the unit of milliradians in the visible wavelength region. On the other hand, in many semiconductor lasers, the beam diameter is in units of microns, and the divergence angle is several tens of milliradians to several hundreds of milliradians.

The configuration shown in FIG. 18 is a configuration suitable for the case where the laser light source 111 has a relatively large beam diameter and a relatively small opening angle in the light emitting portion, such as a gas laser. The laser beam 301 emitted from the laser light source 111 is given a predetermined optical path length through the first optical path adjustment system 410 and then condensed by the condenser lens 401 and collected according to the above-described equation (4). A beam waist is formed near the focal point of the optical lens 401. Further, a beam waist is formed on the conjugate plane in the second camera port 212 by the collimator lens 402. At this time, a beam waist is formed on the conjugate plane in the second camera port 212, and the focal length of the condensing lens 401 and the collimator lens 402 and the condensing lens so that the beam diameter at the beam waist becomes W _port2 described above. It is necessary to adjust the distance between 401 and the collimator lens 402. This adjustment can be performed by changing the optical path length by translating the folding mirrors 412 and 413 in the optical path adjustment system 410. Similarly, the optical path length is adjusted to an appropriate one by moving the folding mirrors 422 and 423 in the optical path adjusting system 420 in parallel.

  As described above, even if the magnification is changed by changing the focal length of the objective lens 102-A, it is not necessary to change the beam waist diameter at the beam waist in the second camera port 212. For this reason, once the focal length of the condenser lens 401, the focal length of the collimator lens 402, the lens interval, and the optical path length described above are set, there is no need to change them thereafter. However, this is not the case when the wavelength of the excitation light source is changed or the beam diameter of the light source light emitting part is changed, such as when the type of fluorescent dye for staining the sample is changed. That is, the focal length of the condensing lens 401, the focal length of the collimator lens 402, and the interval between the condensing lens 401 and the collimator lens 402 are reset appropriately, and the first and second optical path adjustment systems 410 and 420 are readjusted. There is a need to. In order to cope with such a situation, it is preferable that the collimator lens 402 is a variable focal length lens and the condenser lens 401 is movable in the optical axis direction at the same time.

  When the diameter of the light emitting part of the laser light source is small and the divergence angle is relatively large like a semiconductor laser, the beam waist formed at the position of the condenser lens 401 in FIG. 18 is regarded as the light emitting part of the semiconductor laser. The beam waist can be similarly set by using the configuration after the collimator lens 402 as it is. Even when the laser beam is guided by an optical fiber, the configuration after the collimator lens 402 can be used as it is, assuming that the fiber exit is a beam waist formed at the position of the condenser lens 401.

  In the case of using three laser beams, a three-branch optical system shown in FIG. 19 may be used instead of the Mach-Zehnder optical system 430 in FIG. Here, reference numeral 461 denotes a half mirror that divides an incident light beam by an intensity ratio of 1/3 for reflection and 2/3 for transmission, and 462, 465, and 467 are normal half mirrors, and 463, 464, and 466 are the other half mirrors. Is a bending mirror.

  Returning to FIG. 18, modifications necessary for attaching the illumination unit to a normal fluorescence microscope will be described. First, the excitation light filter 201, the dichroic mirror 114, and the fluorescence filter 203 shown in FIG. 17 are removed by rotating the turret or the like. Next, the fluorescent filter 203 or a filter having optical characteristics equivalent to this is installed between the half mirror 204 and the first camera port 211. Thereby, the reflected light 303 by the object 101 of the laser beams 301 -A and 301 -B as excitation light is blocked and prevented from entering the image sensor 103. The fluorescence filter 203 can also be attached immediately before the image sensor 103. This modification is merely a change in the installation position of the fluorescent filter 203 and can be easily performed, and can be easily restored to the original state.

  Next, another configuration for obtaining a plurality of light beams will be described. FIG. 20 is a diagram for explaining an embodiment of a method for dividing a light beam by the diffraction grating 112. In this description, parts different from the embodiment using the Mach-Zehnder optical system already described in FIG. 18 will be mainly described, and description of common parts will be simplified or omitted.

  The illumination unit 400 forms a beam waist on the conjugate plane in the camera port of the second camera port 212, as in the embodiment shown in FIG. However, in this embodiment, the diffraction grating 112 is disposed immediately before the second camera port 212, so that the laser beam 301 is divided into two laser beams 301-A and 301-B. The diffraction grating 112 is constituted by, for example, a thick gradient index diffraction grating. In a refractive index distribution type diffraction grating having a thickness, the refractive index in the element changes three-dimensionally. By optimizing the way the refractive index is distributed, most of the light intensity is equally divided into the 0th-order diffracted light and the 1st-order diffracted light, and the energy distributed to the diffracted light of other diffracted orders such as -1st order is reduced It is known that it can be set to suppress. The diffraction angle of the first-order diffracted light can be adjusted to a desired angle by the pitch of the refractive index distribution.

  There are other possible configurations for dividing the light beam emitted from the light source into a plurality of light beams and making them incident on the second camera port 212. The configuration example shown in FIG. 21 also uses a diffraction grating as the beam splitting means, but is a different type of configuration example from that already described with reference to FIG. A light beam emitted from a laser light source (not shown) enters the optical fiber 452 by a condenser lens (not shown) and is guided to the illumination unit 400. The laser beam 301 diverged from the exit of the optical fiber 452 becomes a substantially parallel beam by the collimator lens 402, enters the diffraction grating 112, and is divided into a zero-order diffracted beam and a first-order diffracted beam. The two light fluxes are collected at the focal plane 113 of the condenser lens 404, and then a second collimator lens 405 forms a beam waist on the conjugate surface in the camera port of the second camera port 212, thereby producing a second camera. A normal fluorescence microscope 200 is entered from the port 212.

  Note that, on the pupil plane 113 of the illumination optical system that constitutes the illumination unit 400, each light beam is condensed at different positions corresponding to the incident angle of the light beam incident on the second camera port 212.

  Here, it is possible to set the diffraction grating 112 and the second camera port 212 to be conjugate with each other by an optical system including the condenser lens and the second collimator lens. By setting in this way, the two separated light beams can be overlapped at the second camera port 212. It is also clear that asymmetric structural illumination can be realized on the object 101 by setting the parallel light beam to enter the diffraction grating 112 at a non-zero angle.

  Next, a method for realizing uniform illumination will be described. Uniform illumination can be realized by using an epi-illumination optical system originally provided in a normal fluorescence microscope. However, in order to construct a three-dimensional image, a large number of sectioning images taken by changing the focus coordinates little by little are required. In order to acquire these multiple sectioning images, frequent switching from asymmetric structural illumination to uniform illumination (or vice versa) must be made. If the illumination unit is removed and the fluorescence microscope is restored to its original state each time the switching occurs, and it is returned to the original epi-illumination optical system, it is very disadvantageous in terms of time. Moreover, if the turret in which the optical filters and the dichroic mirror are stored is frequently operated and these optical elements are replaced, it may cause unnecessary vibration and image displacement.

  Therefore, it is not practical to realize uniform illumination by removing the illumination unit one by one and returning the microscope to its original state. As an alternative means conceivable, the light is shielded except for one of a plurality of light beams so that only the light beam illuminates the object (first method), or a plurality of light beams by utilizing the degree of freedom of polarization. (Second method) and so on. The first method can be realized, for example, by installing a member that blocks the light beam 301 -A in FIG. 18 between the half mirror 431 and the bending mirror 433. The light-shielding member (light-shielding element) can be used with movable equipment that repeatedly inserts and retracts in accordance with the timing of switching between asymmetric structure illumination and uniform illumination. The second method is suitable when a linearly polarized beam is used. For example, by using a polarizing element such as a half-wave plate, if the polarization direction of one of the two divided beams is rotated 90 degrees with respect to the polarization direction of the other beam, these 2 Uniform illumination can be achieved with the two beams no longer interfering with the object plane. Any method for realizing uniform illumination will be described in detail later in an embodiment.

  By using the methods described so far, it is possible to construct a three-dimensional fluorescence microscope system having a high-quality sectioning effect by attaching a lighting unit to a normal fluorescence microscope with simple and easy reversion. . Note that the positional deviation of the pattern of the asymmetric structure illumination does not affect the final image quality as long as the pattern has periodicity. This is because what is required in the computer processing for obtaining the sectioning effect is a standard deviation value of the intensity distribution calculated in the vicinity of each point of the image. Because there is.

  Examples 1 and 2 will be described below for details of a fluorescence microscope provided with an illumination optical system having a pupil function P2, and specific configurations of the illumination unit will be described using Examples 3 to 5.

  Example 1 is an illumination optical system for a microscope having the configuration shown in FIG. 11, and has optical wavelengths of λ = 500 nm and NA = 0.7. O2 is adopted as the object. As shown in FIG. 9A, the pupil function P2 of the illumination optical system is coherent with each other at three vertices of a triangle similar to a triangle formed by the three points represented by the equation (4) in the pupil or the three points. It is expressed as a point light source. In the formula (4), a and b are a = 0.2 and b = 0.1.

  A method of using random speckles disclosed in Non-Patent Documents 5 and 6 for illumination and a method of using lattice illumination formed by the illumination optical system having the pupil function P2 of this embodiment will be compared.

  FIG. 12 shows an image of the object O2 obtained by a method using random speckles for illumination. FIG. 12A shows an xz plane (cross section) when an image is cut along a plane of y = 0 μm. Originally, the image is resolved at the position of z = ± 1 μm where the fluorescent object should be present, but the intensity distribution is non-uniform. FIG. 12B shows an image on the xy plane at z = 1 μm. It can be seen that the illumination unevenness is large overall. FIG. 12C shows a cross section when the image is cut by a straight line of x = y = 0 μm. The intensity is asymmetric in the z direction and the contrast is only about 0.4. FIG. 12D is an image on a straight line with y = 0 μm and z = 1 μm. Intensity non-uniformity is observed.

  On the other hand, FIG. 13 shows an image of the object O2 by the method using the lattice illumination formed by the illumination optical system having the pupil function P2 of the present embodiment. FIG. 13A shows an xz plane (cross section) when an image is cut along a plane of y = 0 μm. Originally, the image is resolved at a position of z = ± 1 μm where the fluorescent object should be present, and the intensity distribution is generally uniform. FIG. 13B shows an image on the xy plane at z = 1 μm. Overall illumination unevenness is suppressed to a low level. FIG. 13C shows a cross section when the image is cut by a straight line of x = y = 0 μm. The intensity is symmetrical in the z direction, and the contrast is sufficiently obtained as 0.7 or more. FIG. 13D is an image on a straight line with y = 0 μm and z = 1 μm. The strength is generally uniform.

  In the first embodiment, the case where the number of point light sources on the pupil plane of the illumination optical system is three has been described. However, in the present invention, the number of point light sources is not necessarily three, and may be two or four or more. But you can. In order to show this, as Example 2, an example in which a sectioning effect is provided using two point light sources will be described.

  FIG. 14A shows the pupil function of the illumination optical system in the present embodiment. Here, the coordinates of the two point light sources on the pupil plane are (0, 0.9) and (0, −0.5). FIG. 14B shows the illumination light intensity distribution formed on the object plane by this pupil function. Such a pupil function can be easily formed by using a diffraction grating or the like as the optical element 112 in FIG. 11 and separating the point light source as the coherent light source 111 into two. As a result, a striped pattern is generated on the object plane by two-beam interference. FIG. 15 shows an image of the object O2 by the method using the lattice illumination formed by the illumination optical system having the pupil function in which the two point light sources are arranged according to the present embodiment. FIG. 15A shows an xz plane (cross section) when an image is cut along a plane of y = 0 μm. Originally, the image is resolved at a position of z = ± 1 μm where the fluorescent object should be present, and the intensity distribution is generally uniform. FIG. 15B shows an image on the xy plane at z = 1 μm. Overall illumination unevenness is suppressed to a low level. FIG. 15C shows a cross section when the image is cut by a straight line of x = y = 0 μm. The intensity is symmetrical in the z direction, and the contrast is sufficiently obtained as 0.7 or more. FIG. 15D is an image on a straight line with y = 0 μm and z = 1 μm. The strength is generally uniform.

  Thus, in the present invention, the number of light source regions is not limited as long as it is two or more (plural).

  The time required for processing when imaging with 256 × 256 × 256 pixels using a three-dimensional microscope using the illumination optical system of Examples 1 and 2 is 1 minute on a workstation equipped with a 3.33 GHz CPU. Is within. By creating a dedicated program and optimizing and using hardware devices such as a parallel distributed environment and a graphic accelerator, it is possible to obtain an image in a time shorter than the time required for scanning by the confocal microscope.

In the third embodiment, using numerical values (specified values) such as the magnification of the microscope shown below, numerical examples relating to the setting of the illumination area and the setting of the beam waist and beam diameter are presented, and the configuration shown in FIG. Indicates that it can be implemented.
-The length of half of the diagonal of the image sensor 103: W _image = 4 mm
-Focal length of the objective lens 102-A: _fobj = 4mm
-Focal length of imaging lens 102-B: f _tube = 160mm
Imaging magnification from the object 101 to the image sensor 103: m = 40 times Wavelength of the laser light source 101: λ = 488 nm
-Focal length of condensing lens 401: f ₁ = 15 mm
-Radius of laser light source 111 light emitting part: w _o = 0.26 mm
In Table 1 shown below, the unit of the optical parameters excluding the wavelength of the laser light source 101 is mm. When the specified values are determined as described above, the values of other optical parameters are calculated using these specified values. In the following, it is shown that the values of the other optical parameters are realistic values. Numerical examples used in this embodiment are summarized in Table 1. The upper part of the table shows the prescribed values, and the lower part of the table shows the values of the following parameters determined from the prescribed values.
Optical path length from the laser light source 111 to the condenser lens 401 via the first optical path adjustment system 410: d ₀
A beam diameter of a beam waist generated near the focal point of the condenser lens 401: w ₁
-Focal length of collimator lens with variable focal length: f ₂
The distance between the collecting lens 401 and the beam waist generated near the focal point of the collecting lens 401: d ₁
The distance between the beam waist generated near the focal point of the condenser lens 401 and the collimator lens 402: d ₂
The optical path length from the collimator lens 402 to the conjugate plane in the camera port of the second camera port 212 through the optical path adjustment system 420 and the Mach-Zehnder system 430: d ₃
The beam diameter of the beam waist generated on the conjugate plane in the camera port of the second camera port 212: W _port2
Now, setting the distance _{d 2} between the condenser lens 401 and the collimator lens 402 to 98.7754mm + 15.2mm. At this time, by adjusting the optical path length _{d 0} extending from the laser light source 111 emitting portion to the first condenser lens 401 via the optical path adjusting system 410 933.898Mm, as expected area asymmetric structure illumination is made, _{W image} = 4 mm.

Note that a general mount such as a C mount or a T mount is often used for a connection portion between the illumination unit 400 and the second camera port 212 in FIG. For example, when a C mount is used for connection, the attachment error in the direction perpendicular to the optical axis is about ± 0.1 mm. In the present embodiment, the area where the asymmetric structure illumination is performed is defined as W _image = 4 mm, and the attachment error with respect to this value is about 2.5%. When an attachment error in a direction perpendicular to the optical axis of the C mount occurs, the region captured by the image sensor 103 on the object 101 and the region subjected to asymmetric structure illumination are shifted in the horizontal direction. However, there is usually no problem if a lighting failure occurs in an area of about 2.5% outside the image. On the other hand, there is a possibility that an error in the direction of the optical axis occurs when joining by mounting. Now, it is assumed that the deviation in the optical axis direction is about ± 0.1 mm. When calculated by the equation (4), due to this error, the deviation in the optical axis direction between the object plane that is the focal point of the objective lens 102-A and the beam waist formed there is about 63 nm. Since this value is sufficiently smaller than the depth of focus of the objective lens 102-A, it can be ignored.

  In the present embodiment, in the configuration described with reference to FIG. 18, among the plurality of light beams used for forming the asymmetric structure illumination, one light beam is left and the other light beams are shielded, and as a result, only one light beam illuminates the object. On how to do. Here, an example will be described with reference to FIG. A plurality of light fluxes generated by the diffraction grating 112 are condensed on the pupil 113 of the illumination optical system by the lens 404 to form a plurality of spots. The pupil 113 of the illumination optical system has a conjugate relationship with the pupil of the objective lens 102-A. Therefore, if a light shielding mask 501 as shown in FIG. 22 is arranged in the vicinity of the pupil 113 of the illumination optical system or its conjugate part, the light transmittance of a plurality of spots can be controlled.

  FIG. 22A shows an example of the structure of the light shielding mask 501 suitable for the case where the plurality of light beams are composed of two and FIG. 22B is the case where the plurality of light beams are composed of three. is there.

  In FIG. 22, among the plurality of spots, the spots to be shielded are indicated as 502-A and 502 -B, and the light shielding portions 503-A and 503-B arranged in a part of the light shielding mask 501 are moved. The spots to be shielded can shield 502-A and 502-B. In this way, only the light beam corresponding to the remaining one spot is irradiated onto the object surface, and uniform illumination is realized. The movement of the light shielding portion can be easily performed by rotating a disk-shaped light shielding mask 501 around the optical axis, for example. If it is necessary to perform asymmetric structure illumination again, the light shielding mask 501 may be moved again to return to a state where a plurality of spots are not shielded.

  The moving method of the light shielding unit is not limited to rotation around the optical axis, and any method can be used as long as the operation described above is realized. For example, the light shielding mask 501 may be slid in and out of the optical path. Moreover, the drive system is not ask | required. When a large number of light-shielding portions 503 having a small area are arranged on the light-shielding mask 501, it is possible to switch between asymmetric structure illumination and uniform illumination mode by moving the light-shielding mask 501 only slightly.

  The light shielding unit 503 can be realized by using a member having a very low transmittance with respect to general light, or using a polarizing element that selectively blocks light in a specific polarization state. Furthermore, if a liquid crystal polarizing plate or the like that can dynamically change element characteristics such as light transmittance by an electrical signal is used as the light shielding portion 503, it is not necessary to move the light shielding mask 501.

  In this embodiment, a configuration example of an optical system is provided that includes a branching unit for dividing a beam into a plurality of light beams and realizes switching between asymmetric structure illumination and uniform illumination by controlling the polarization of each light beam. FIG. 23 is a schematic view showing such a configuration. The configuration of the illumination optical system illustrated in FIG. 23A has many similarities to the illumination optical system illustrated in FIG. 21, and thus description of the common configuration is omitted, and the second camera port 212 from the light source is omitted. Only the optical path configuration up to now will be described. Light emitted from a light source (not shown) is guided to the collimator lens 402 through the fiber 452 and converted into a parallel light beam. The light source considered here is a coherent light source, and is linearly polarized in a direction perpendicular to the paper surface. The polarization shaping element 471 is, for example, a half-wave plate, and if the optical axis is inclined at 45 ° with respect to the polarization direction of the parallel light flux, the parallel light flux is converted into a linear polarization component in the direction perpendicular to the paper surface. (S wave) and linearly polarized light component (p wave) in the depth direction of the drawing.

  Next, the configuration of the beam interference optical system 430 including the elements 472, 473, and 474 will be described. The s wave is reflected by the polarization beam splitter 472. On the other hand, the p wave passes through the polarization beam splitter 472. The s-wave reflected by the polarization beam splitter 472 propagates directly to the second camera port 212, but the p-wave transmitted through the polarization beam splitter 472 goes to the bending mirror 473, where it is redirected. The p-wave whose direction has been changed is converted into an s-wave by a half-wave plate 474 having an axis perpendicular to the optical axis and inclined by 45 ° from the p-wave polarization direction, and then reaches the second camera port 212. Since the above two light fluxes are both s waves at the position of the second camera port 212, they have coherence on the second camera port 212 and the object 101, and asymmetric structure illumination can be formed.

  On the other hand, in order to realize uniform illumination, the polarization state after passing through the polarization forming element 471 may be set to be only s waves or only p waves. Then, only one s wave or p wave beam reaches the second camera port 212, and uniform illumination can be formed. In addition, in FIG. 23, the description has been given on the assumption that the asymmetric structure illumination is generated by the two-beam interference, but it is also possible to generate an interference of three or more beams by adding a beam splitting unit by polarization. Further, it is optional to appropriately add a polarizing element to the optical path from the time when the plurality of light beams are branched to the time when each light beam reaches the second camera port 212, and to adjust the polarization state of each light beam to an optimum one. is there.

  The sine of the angle θ between the two light beams shown in FIG. 23A is inversely proportional to the lateral magnification β of the second camera port 212 with respect to the object 101 on the conjugate plane in the camera port. More specifically, the numerical aperture of the objective lens 102-A is NA, the radius of the pupil corresponding to NA is r, the refractive index of the medium on the sample surface is n, and the distance between spots on the pupil 113 of the illumination optical system is d. Then, the sine of the angle θ is expressed by Expression (7).

As an example, if NA = 0.95, d / r = 1, and β = 40, sin θ = 0.024, and the angle θ becomes considerably small. At this time, in order to avoid physical interference, if an attempt is made to secure, for example, a distance of 5 cm between the polarizing beam splitter 472 and the bending mirror 473, the distance between these two elements and the conjugate plane in the camera port of the second camera port 212. Needs about 2m. This impairs the compactness of the lighting unit. In order to solve this problem, as shown in FIG. 23B, a beam expander 476 (magnification m _exp ) is inserted into the beam interference optical system 430, and the angle formed by the two light beams is expanded to m _exp times. That's fine. This makes it possible to configure a lighting unit having a practical size.

  Each embodiment described above is only a representative example, and various modifications and changes can be made to each embodiment in carrying out the present invention.

  An illumination optical system that can be used in a microscope such as a fluorescent microscope or a digital slide scanner can be provided.

110 Illumination optical system 111 Coherent light source 113 Pupil (lens) of illumination optical system

Claims (14)

An illumination optical system for illuminating a sample placed on an object plane,
A plurality of coherent light source regions are arranged apart from each other on the pupil plane of the illumination optical system,
An illumination optical system characterized in that at least one of the distances between the centers of the plurality of light source regions and the pupil center of the illumination optical system is different from other distances.   The illumination optical system according to claim 1, wherein the sample is a self-luminous body.   The illumination optical system according to claim 2, wherein a light emission mechanism of the sample which is the self-luminous body is fluorescence or phosphorescence. The illumination optical system according to any one of claims 1 to 3,
A microscope having a projection optical system for forming an image of the sample. A first camera port and a second camera port disposed at a position optically conjugate with the object plane;
The first camera port includes an image sensor for imaging the sample,
The second camera port allows the plurality of coherent plural light beams having a size ratio of less than 0.3 to a pupil radius of the illumination optical system to be incident on a plurality of collimated light beams that are coherent to each other. The microscope according to claim 4, wherein the light source region is formed.   The second camera port includes a plurality of coherent parallel beams at a position optically conjugate with the object plane so that the plurality of coherent parallel light beams form a beam waist and overlap each other. The microscope according to claim 5, wherein a light beam is incident on the object plane with an incident angle.   Excitation light is blocked between the first camera port and a position where an optical path from the object plane to the second camera port and an optical path from the object plane to the first camera port branch. The microscope according to claim 5 or 6, further comprising an optical element that transmits fluorescence or phosphorescence.   The movable light-shielding element for switching between a state in which at least one of the plurality of parallel light beams coherent with each other is shielded and a state in which none of the light beams is shielded is provided. The microscope according to one item.   The microscope according to claim 8, wherein the light shielding element shields light from a wavelength of fluorescence or phosphorescence.   The microscope according to claim 8 or 9, wherein the light shielding element includes at least one polarizing element.   The microscope according to any one of claims 5 to 10, further comprising a mechanism for controlling coherence of the plurality of parallel light beams that are coherent with each other.   The microscope according to claim 10, further comprising a mechanism for adjusting a polarization direction of the plurality of parallel light beams that are coherent with each other by the at least one polarizing element.   The microscope according to claim 5, wherein the microscope is a fluorescence microscope. The microscope according to any one of claims 5 to 13, wherein the microscope is an episcopic microscope or a transmission microscope.